FR2635587A1 - APPARATUS AND METHOD FOR OPTICAL DENSITY MEASUREMENTS OF BIOMASSES - Google Patents

Abstract

The invention relates to an optical density measuring apparatus. This apparatus comprises an optical probe 2 with optical fibers 12, 13, with a detection cell 6, a light source 14 and a light detector 16 to which are connected means for linearization of the output signal, interference compensation means caused by agitation and gas bubbles, and means for recording the results. Application: advanced methods for direct real-time monitoring of cell density.

Description

The present invention relates to an improvement

  relating to or described an apparatus and a method for obtaining optical density measurements as a means of controlling with respect to a system of measurement; dynamic transformation of biomass, such as a fermentation process, anaerobic or aerobic, of microorganisms. The present invention more specifically relates to a method for determining the direct optical density of a control system.

  dynamic processing in the entire operational range

  by compensating for the primary interference factor.

  The measurement of cell density is important in the control of the program of a

  fermentation. Many processes and apparatus correspond to

  have been proposed for the measurement of cell density. None of the prior methods and apparatus have been found to be successful and satisfactory prior to the present invention. Preferably, the use of a sterilizable probe, which can be inserted directly into the fermenter or nutrient medium for measurement of cell density, would be advantageous and would provide valuable information in controlling the evolution of the process. Usually the product concentration is related to the cell mass whose determination can be

  obtained from measurements of cell density.

  So far, the cell density has been

  measured in discrete intervals by taking samples

  in the fermentor. This procedure does not allow

  It was not a matter of obtaining a direct and rapid dosage of the product, but resulted in an estimate of the cell mass which is an indication of the concentration of product at the time of sampling in the fermenter. To achieve a satisfactory and precise operational control of a direct measurement system, a

  Continuous measurement system is desirable.

  Indirect control measures are based on different operating parameters and are limited by

  the precision of the mathematical model of cell growth

  of substrate consumption and formation of

  product. The assumptions concerning the returns

  tants, the holding coefficients and the stoichiometry may not be valid throughout the entire operating range due to a depletion of substrates and an accumulation of intermediate substances that can be metabolized, as well as product formation.

inhibitors.

  The method of measuring discrete interval cell density is at best a proxy density measurement technique. From a sterilized sample collection port, a small volume of culture broth is taken after ensuring that the orifice has been purged. The sample is transported to the bench of the laboratory and the appropriate dilution of the sample is carried out. Particular care must be taken in the accuracy and regularity of the pipetting technique and the equipment, as well as those of other operators also taking samples at intervals. Finally, the optical density is measured, multiplied by the dilution factor and the value is recorded. During this time, from taking the sample to recording the measured value, it is not known which next appropriate operating step

  will be necessary to optimize the overall process.

  The method of measuring the cell density at discrete intervals is not likely to be automated. One must rely on any parameter measured indirectly: metabolic or physical. Measurements in dynamic systems have different disadvantages. The physical complexity of the cultures of

  microbial fermentation is influenced by the characteristics

  middle ticks, dimensions, shape and type of the body; operating variations: pH, temperature, pressure, agitation, etc. The metabolic complexity of growing crops is influenced by

  microbial growth phases in response to the en--

  physical environment. This affects cell size, replication duplexes, chain formation, inclusions, corpuscular formation, etc. Similarly, for microorganisms growing in a complex nutrient broth, there is no certainty about the nutrients that are used at each stage of development in this process. As a result, he

  is difficult to prepare a growth model.

  Until now, optical sensors have been advantageous for measuring the amount of light passing through an operating fluid. However, the amount of light transmitted through any particular operating fluid can be reduced by various factors such as suspended solids, dissolved solids, and emulsion formation. Suspended solids and emulsions also reduce the light transmittance of light scattering. In an aerobic environment, the optical density appears to be influenced by the number of bubbles and the dimensions of these bubbles. Large gas bubbles, such as air bubbles, scatter the light in exactly the same way as large particles, but retain some light transparency. At high stirring speeds, when smaller bubbles are produced, the dispersion of the light has a reduced effect and the transparency is increased. As a result, a biomass system poses unique problems associated with measurements by means of optical sensors. Accordingly, the object of the present invention is to provide an apparatus and method for direct optical density measurement of a dynamic biomass processing system in which organisms are cultured, direct rapid measurements of cell density. being required; the measurements are carried out continuously or in real time relative to the process; On-the-spot measurement is used to avoid problems with successive discrete samples such as interval sample logging, sterilization, disruption of the process, time between sampling of a sample and the recording of the measurement, the rapid determination of the operative step

  appropriate when the sample is taken.

  Another object of the present invention

  consists in proposing a method for determining the con-

  concentration of an aerobic or anaerobic biomass system in

  terms of offsetting any international factor

  the optical density, such as gas bubbles, which ensures linearity throughout the measurement range. Another objective is to provide an optical density measuring device which facilitates the continuous, real-time automatic control of the

  fermentation process, resulting in an automatic

total process.

  A general objective of the present invention

  is to overcome the disadvantages of the prior art.

  Still other objectives will appear below in the

following detailed description.

  These objectives are achieved in the present invention by using a fiber optic sterilizable probe having an opening or a cell path distance through which a fluid containing a biomass reacts, and having a light source for optical fibers and a light source. light sensor transmitted by optical fibers. In operation, the light from the optical fiber constituting the light source passes through the medium of the sample into the aperture of the cell and the transmitted light is then passed through the receiving end of the fiber optic conduit and sent to the transmitter; said transmitter consists of a signal amplifier and a computer having a program for linearizing the output signal. The optical probe is removably enclosed in a fermentor or similar element provided with

  control instrumentation.-

  In general, the optical sensor in the probe measures the amount of light that passes through an operating fluid present in the opening of the cell. The system is able to automatically compensate the

  presence of gas bubbles in the opening of the cell.

  Other features and benefits of the

  present invention will emerge from the detailed description

  which follows, made with reference to the accompanying drawings in which: Figure 1 is a schematic diagram of the entire operating system; Figure 2 is a schematic representation of the probe with the measuring cell; FIG. 3 is a schematic representation

  reactor-fermentor for biological systems.

  With regard to the drawings, and in particular

  In particular Figure 2, reference numeral 2 denotes a probe according to the present invention. The probe 2 may be made of any material not subject to corrosion by the medium present in the installation. However, the probe 2 is preferably made of a stainless steel case. The probe consists of an Ingold 4 connector which allows the insertion of the probe 2 into the body or the wall of a reactor 1, such as a fermentation tank, and a fastening thereto in a firm and removable manner. The connector Ingold 4 and an O-ring device 5 allow the probe 2 to be tightly fitted through an access tube 3 connected to the wall of the reactor 1 and the access tube 3 has a connection

  adapting to that of the Ingold 4 fitting.

  Inside the probe 2 is a radiation light source 14 and a sensitive photodetector 16. The light source 14 and the detector 16 are each connected respectively to electrical conductors 17 and 18. The light source 14 is The detector 16 transmits a signal pulse through the current conductor 18 to a signal amplification and recording means. The radiation light source 14 and the photodetector 16 are separated from the operating medium by the

  optical fibers 12 and 13 which are used for transmitting

  the light 12 of the source and the light 13 detected.

  The cell orifice at the end of the probe protruding into the reactor and the reaction medium therein, it is defined by a path length a sampling space 6 in which the optical fibers 12 and 13 are terminate with windows 10 and 11 hermetically fixed. The windows 10 and 11 are the end portions of the respective optical fibers 12 and 13 and have

  for the role of protecting the ends of the optical fibers.

  The windows 10 and 11 are generally placed facing each other in the sampling space 6. In addition, these windows 10 and 11 may be made of any light transmitting material, which allows the required light radiation to pass through the sampling space 6 and, similarly, which allows the detection of the light transmitted through the material present in the sampling cell 6. Preferably, the windows are made of Pyrex or

quartz.

  The pickup optical fiber 13 is connected to a photodetector 16 which is in turn connected to a current conductor 18. The photodetector is covered by an optical filter 15 which eliminates all light wavelengths less than 850 nanometers, suppressing thus interference by ambient light. The wavelength cutoff threshold of the filter thus eliminates the interference caused by a color. As a result, most particles appear similar at this wavelength

high.

  During the implementation of the method, the solution in the fermenter is first a substantially clear broth and is transformed during the implementation of the process into a very dark color solution. An optimum optical path length is chosen in the sampling space 6 to allow the

  probe an examination throughout the operating range.

  The measurement interval is inversely proportional to the path length and the resolution is directly proportional to the path length. Consequently, the shorter the path length in the cell 6 of the optical probe, the larger the observable range,

  but the lower the resolution.

  The classical unit of measurement is the absorption unit (U.A.). This is an arbitrary unit that is defined when used in the following manner: U.A. is the amount of light absorbed in a clear nutrient medium in the absence of cell growth. Each additional unit corresponds to a decimal variation (factor 10, ie the logarithm of the variation) of the amount of light absorbed and is correlated to the intensity of the output current by the

photodétecteur.-

  A problem with such a system is that it is possible that the correlation between the readings

  of light absorption and the actual measurement of

  tion is not linear throughout the entire range.

  -In an extremely small segment of the absorption curve

  There is a linear relationship between the process and the output light signal. But, in the case of a measurement in a wide range, there is no linear relation, which means that a variation in a limpid region does not correspond to an equal or equivalent variation in the dark region. However, this imbalance or unequal variation may be linearized in the programmed transmitter of Figure 2 and Figure 3. The transmitter is a microprocessor-controlled transmitter with a digital keypad input source and a controller. character display. Consequently, by understanding the behavioral relations between particles and knowing a certain phenomenon concerning the nature of the light in relation to the concentration, it is possible to extrapolate a precise linearized curve from

only two entry points.

  When a conventional microbial fermenter equipped with the necessary control instrumentation has been used, the internal device inside the fermenter consists of a series of radially oriented baffles and a heat exchange unit. Baffles prevent a

  swirling to improve the efficiency of the aerator

  tion. Also used is a means for effectively dispersing small bubbles in the medium, such as three disc turbine stirrers placed on the fermentor agitator shaft. A mixed spray orifice / agitator device (perforated conduit) produces and disperses small air bubbles throughout the entire

  fermenter. The stirring speed, which can be variable

  adjusted to 1000 rpm, has been set in

tor.

  The stirring has the role of increasing the available surface area for the transfer of oxygen by dispersing the air in the culture broth in the form of small bubbles. Stirring also delays the escape of air bubbles, i.e. increases the contact time, in the liquid and prevents cales. Stirring decreases the thickness of the liquid film at the gas / liquid interface by creating turbulence in the culture zone. In general, this apparatus, as described, is very effective in producing a large number of very

  small bubbles of gas in the fermenter.

  In the aerobic fermentation system defined above, agitation speed (turbulence) is the main interference factor in obtaining

  precise and representative measurements of the cell density

  lulaire. Airflow and air pressure have little effect. The problem of uncontrolled foam formation is caused by increased residence time of the bubbles. As foam formation continues, there is a quantitative increase in

diameter and number of bubbles.

  The stainless steel probe is attached

  Removable and waterproof way through the wall of the fermen-

  tor, so that the sensitive part of the probe containing the optical fiber of the light source, the optical fiber of the photodetector and the density measuring cell is directly introduced into the fermentation medium. As the fermentation progresses, the liquid medium can go from a virtually clear solution to a very opaque solution. An optical path is chosen in the probe to enable the probe to be detected throughout the entire operating range. In the case of measurements in a wide range, the correlation between the values

  observed and actual measured values may not be linear.

  This means that a variation in the clear solution does not match or indicate equal variation in the opaque region. As a result, the probe is coupled to a transmitter that is programmed to linearize the measurements

in a wide range.

  It has been found that the agitation rate has an effect on the observed direct cell density. The signal variation from the probe was found to be proportional to the stirring rate and inversely proportional to the cell density. As a result, the probe signal is a composite value which depends on: a) the cell density and viscosity of the solution, b) the stirring speed if it is greater than 400 rpm, and c) diameter and number of

bubbles in the liquid medium.

  The correction model, which adjusts the raw density results, is based on two linear equations. Each equation correlates the direct cell density value with the density

  optical at a particular stirring speed (in rpm).

  The intersection between the linear equations is a point where the agitation has no effect on the signal. This means that, below this point, an increase in agitation makes the appearance of the solution darker, that is to say more opaque (higher optical density), under the influence of the number of bubbles and their diameter; and, above this point, an increase in agitation makes the appearance of the solution more clear (lower optical density). The phenomenon is attributed to the effect of bubbles in the medium on the total signal of the probe. This means that at this point the transparency effect is equal to the scattering effect due to the presence of bubbles

in the fermentation medium.

  Another factor introduced into the equations

  of linearisation is the known effect of the stirring speed

  on the bubbles. It has been found that at a point other than the point of intersection there is a non-linear correlation between the stirring speed (in rpm) and the signal variations. It is chosen an equation that best approximates the phenomenon between 200 tr / uin and

  1000 rpm. Low or no aeration occurs

  below 200 rpm and, similarly, low or no aeration occurs above 1000 rpm, the aeration process starting to stop at a certain level due to the design limitations of the

  general process and equipment.

  It has been found that the signal from the probe is a factor in the stirring speed. The answer also depends on the ratio of cell concentrations

  to air bubbles, compared to the stirring speed.

  This means that, under the same conditions, at low cell densities, the signal is stronger at high stirring speeds than it is at high cell densities. A curve in the region of weak agitation

  was chosen for the greatest precision in the inter-

  which was the most important for the fermentation process. A polynomial derivation of the curve was determined from the results collected from the effect of the stirring rate on signal power at a given or chosen optical density. The curve was in the form: Pouzentage of density variation cptique = ko + kAg + k + k3Ag

  k being a constant and Ag being the stirring speed.

  The transmitter generates a response curve that closely approximates the phenomenon, that is, a linear variation in the concentration range, and adjusts the curve to fit the actual operating fluid. The transmitter generates the response curve by performing direct readings or in sampling vessels, storing the readings and the stored readings are assigned values

  which have been determined in the laboratory.

  The instrumental system uses a programmed computer that measures the concentration or consistency of a fluid by absorbing light. The programmed computer monitors a process continuously and generates a value consistent with the process calibration curves. At a minimum, a two-point calibration curve is required to linearize the output signal. Thus, indirect sampling and laboratory errors are reduced and the result is representative of the

  concentration of the fluid in real time.

  The programmed computer is a modular microprocessor-based digital instrument consisting of printed circuit boards, analog output boards, a central processing board for program execution, which includes electronics and interprets the measurement results, a digital communication plate, a

  front platinum plate for the purpose of communica-

  with the man, a lamp output wafer for adjusting the proper voltage for the detection lamps with network voltage loss compensation; a motherboard for interconnecting all the wafers to form the integrated instrumental system. A power supply provides stabilized and unstabilized power to the circuits, relays and sensors; a sensor input pad that conditions a sensor signal to standardize

  the input for the readability of the microprocessor.

  During operation, the microprocessor can interpret and display measurements from different sensors. In general, optical sensors measure the amount of light that passes through an operating fluid. The amount of light transmitted can be reduced by suspended solids, dissolved solids, and emulsions of the type described above. Suspended solids and emulsions also reduce the

transmittance of light.

  As mentioned above, the conventional measurement unit is the U.A unit. arbitrary (absorption unit). The

  U.ä. is defined when using as: U.A.

  the amount of light absorbed in a clear solution (or zero solution). Each additional unit is a decimal variation of the amount of light absorbed and, therefore, can be correlated to the current output by the photodetector. For example, zero U.A. can

  corresponding to 10 mA of current output by the photode-

  1 U.A. would then correspond to 1 mA and 2 U.A.

  would correspond to 0.1 mA output, etc. The problem that the present invention overcomes is the correlation between brightness readings and actual cell density measurements, which may not be linear. In any small segment of the resulting curve, there is a linear relationship between the operating cell density and the output light power. But, in a wider range, there is no linear relationship; the behavior of particles in clear media does not

  has no correlation with a behavioral variation

  equivalent in the obscure or opaque region. This is linearized in the transmitter by the programmed microprocessor.

  Thus, the disadvantages of prior

  are removed. An improved direct estimation system results in more accurate and rapid determinations of cell density in fermentors and similar reactors. Even in the turbulent environment of a fermenter, with the presence of bubbles from the aeration of the reaction mixture present in the reactor, it has been possible with the present invention to resolve cell densities greater than 100 DC and to couple the system directly to a computer for

the operative control.

  It is understood that each of the elements described above, or two or more of these elements, may also find a useful application in other types of constructions, devices, etc., which differ.

types described above.

  Although the present invention has been illustrated and described in an embodiment for

  to measure the turbidity of a liquid in which micro-

  organisms are in culture, it is not intended to be limited to the details shown and described, since different modifications and structural variations can be made without departing in any way from

  the spirit and scope of the present invention.

  Without further analysis, the foregoing reveals the essential object of the present invention, an object which the person skilled in the art can easily adapt, using his usual knowledge, to different applications, without omitting features which, from the point of view of the prior art, constitute the essential characteristics of

  the general or particular aspect of the present invention.

Claims (8)

  A method for controlling a dynamic biological system in a biological reactor containing a growing culture fluid medium, by measuring the light transmitted in a defined sample cell, characterized in that it comprises the steps of to perform simultaneously: a) the irradiation of the fluid medium present in the cell, b) the continuous stirring of the fluid culture medium, c) the detection of the transmitted light, d) the transformation of the detected transmitted light into a signal nonlinear electrical output, e) linearization of said nonlinear output electrical signal, f) compensation of the effect of agitation on said linearized output signal, and g) the recording of the signal.   2. Method according to claim 1, characterized in that a control of a dynamic biological system is carried out by measuring a direct optical density of the present growing fluid culture medium. in the cell.   Apparatus for measuring the direct optical density of a liquid biological medium in a biological reactor in which said liquid medium is agitated, said liquid medium containing a developing and growing biological organism, characterized in that it comprises means for introducing an optical probe (2) comprising a sensing cell (6) into   said liquid biological medium, said probe (2) comprises   taking a light source (14) and a light detector (16) positioned opposite one another in said cell (6), and comprising means connected to said light detector (16) and outside said reactor, for linearizing the output signal of said detector (16) and means for compensating for interference caused by agitation of the liquid medium in the reactor on said output signal; as well as means for recording   optical density results in the cell.   4. Apparatus according to claim 3, characterized in that the means for introducing the probe (2) comprising an optical pickup cell (6) in the   liquid biological medium consists of a   taking optical fibers (12,13) carrying the light to the cell and, in a position opposite to the cell, a photosensitive detector (16) connected to the optical fibers (12,13) for conveying the signal to an amplifier and a outdoor recorder.   5. Method for determining the cell mass   of a biological system by performing an adjustment calculation   Curve of optical density and agitation results obtained from a biological reactor containing a growing culture fluid, characterized in that it comprises the steps of: selecting at least two sets of velocities of shaking and corresponding optical densities, linearizing the results in a transmitter by a programmed microprocessor and defining the value of the cell mass as a function of the stirring and the optical density and to compensate for the effect of the stirring by   two linear equations each of which correlates   between the continuous cell density value and the optical density at a particular stirring speed, thereby establishing a relationship between the mass cell and optical density.   6. The process of claim 5   characterized in that the linearization is the determination   at least two slopes of said linear equations for the linear relation between two series of at least two points each for the stirring speeds and the density corresponding optical system.   7. A method according to claim 6, characterized in that the linearization step is based on two distinct points, at least twenty percent apart from the instrumental operating range and different from zero, where zero represents a base fluid in the process. biological reactor. 8. Process according to Claim 5, characterized in that a stirring speed is chosen in the form of a constant value determined by the oxygen requirement of the cells in culture in the reactor.   biological composition containing the culture fluid.